Properties of tRNA species modified in the 3'-terminal ribose moiety in an eukaryotic ribosomal system

Abstract

Phe-tRNAPhe species [from yeast] modified on the 3''-terminal ribose residue were investigated for their ability to participate in individual steps of the elongation cycle using eukaryotic ribosomes from [rabbit] reticulocytes. None of the Phe-tRNAs used, namely Phe-tRNAPhe-C-C3''dA, Phe-tRNAPhe-C-C-3''-NH2A, and Phe-tRNAPhe-C-C-Aoxi-red, can function in the overall process. All modified Phe-tRNAPhe species can be bound nonenzymatically to ribosomes. Phe-tRNAPhe-C-C-3''NH2A exhibits exceptionally high binding at low Mg2+ concentration compared with Phe-tRNAPhe-C-C-A binding. Ac-Phe-tRNAPhe species prepared from the 3 modified tRNAs, when bound to the donor site, were devoid of donor activity. The enzymatic binding of both Phe-tRNAPhe-C-C-3''dA and Phe-tRNAPhe-C-C-3''NH2A is less efficient than that of Phe-tRNAPhe-C-C-A but these Phe-tRNAPhe species have acceptor activity. Phe-tRNAPhe-C-C-Aoxi-red is not a substrate for the [rat liver] EF-I promoted binding reaction and has no acceptor activity. The nonaminoacylated species, tRNAPhe-C-C-A, tRNAPhe-C-C-3''dA, and tRNAPhe-C-C-3''-NH2A, bind to the ribosome to a larger extent than the corresponding aminoacylated tRNAs, both in the presence and in the absence of poly(U). Peptidyl-tRNAPhe-C-C-3''dA bound to the donor site cannot activate the acceptor site for EF-I promoted binding of Phe-tRNAPhe as does peptidyl-tRNAPhe-C-C-A. Further, it was observed that a correct codon-anticodon interaction influences the recognition of the 3'' terminus of tRNA. Specificity of eukaryotic ribosomes for the 2''- and/or 3''-aminoacylated tRNA species is discussed and compared with the properties of Escherichia coli system.